Chromosome segregation in eukaryotic cells is mediated by the mitotic spindle, a complex machine comprised of microtubules and associated proteins. Our long term goal is to understand the molecular basis of spindle assembly and chromosome segregation. We have identified two microtubule-binding proteins that play essential roles in these processes in the yeast, Saccharomyces cerevisiae. Stu1p is a component of the mitotic spindle and is required for assembly of a bipolar spindle. Stu2p is a component of the spindle pole body and is required for spindle elongation. In addition, we identified three proteins, Spi6p, Bim1p and Bik1p, that interact with Stu2p in vivo. The aims of this proposal are to answer the following questions: 1. What is the nature of the Stu2p complex in yeast? We will determine whether Stu2p, Spi6p, Bim1p and Bik1p reside together in a single unique complex, or whether Stu2p forms separate complexes with these proteins. We will also determine the complete subunit composition of the Stu2p complexes and characterize the interactions among these subunits. 2. What are the cellular roles of Stu2p complex components? We will create mutations in the genes encoding these proteins and examine the phenotypes of cells containing these mutations. These studies will allow us to determine how each mutation affects spindle function, the assembly of microtubule structures, and the dynamics of microtubules in vivo. 3. Are the Stu2p complexes cell cycle regulated? We will determine whether levels of the Stu2p complex proteins and assembly of the Stu2p complexes are cell cycle regulated. In addition, we will examine whether Stu2p complex proteins are phosphorylated and, if so, whether phosphorylation is cell cycle regulated. 4. Which proteins interact with Stu1p? As a component of the mitotic spindle, Stu1p likely interacts with a variety of other spindle components. The identification and characterization of these proteins is essential to understand how Stu1p functions. We will employ both genetic and biochemical approaches to identify proteins that interact with Stu1p and specify their roles in spindle function.